Biomarkers of Microcirculatory Dysfunction in Sepsis: A Pilot Prospective Observational Study.

CONTEXT: To evaluate and compare sublingual microscopy (SLM) to urinary glycosaminoglycan (GAG) assays, dimethylmethylene blue (DMMB) assay, and liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of GAGs as biomarkers of microvascular dysfunction in patients with sepsis and septic shock. HYPOTHESIS: Indicators of microvascular dysfunction and markers of endothelial glycocalyx (eGC) degradation would be associated with sepsis. METHODS AND MODELS: Prospective, observational case-control study. SETTING: Denver Health Medical Center, a safety-net hospital in Denver, CO. SUBJECTS: Forty-four adult patients with sepsis or septic shock and 24 healthy control patients undergoing elective orthopedic procedures. Exclusion criteria included pregnancy and incarceration. RESULTS: Sublingual microvascular parameters (De Backer Density, proportion of perfused vessels) were measured using darkfield sidestream microscopy, and urinary GAGs were measured via DMMB colorimetric assay and LC-MS/MS targeting heparan sulfate (HS), dermatan sulfate, and keratan sulfate. Validation of HS quantification was performed on a subset using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS). LC-MS/MS HS was significantly higher in sepsis vs. controls (Area under the curve 0.85; 95% CI, 0.76-0.95), demonstrating higher diagnostic performance than SLM (De Backer Density AUC 0.71) and DMMB GAGs (AUC 0.62). LC-MS/MS and HILIC-MS HS levels were strongly correlated (R² = 0.97, p < 0.001). DMMB GAGs were associated with HS subtypes (p = 0.05) and SLM density (p = 0.03). No significant associations with in-hospital mortality or acute kidney injury were observed. INTERPRETATION AND CONCLUSIONS: Among the evaluated modalities, LC-MS/MS quantification of HS showed the greatest discriminative ability for identifying sepsis and correlated strongly with established mass spectrometric methods. SLM exhibited moderate diagnostic utility and significant associations with GAG levels, reinforcing its biologic relevance. However, its bedside application may be limited by challenges in image acquisition and analysis. The concordance across SLM, DMMB, and LC-MS/MS supports eGC degradation as a key feature of sepsis pathophysiology. These findings highlight the promise of LC-MS/MS as a scalable, rapid, and mechanistically informed platform for biomarker-driven enrichment in future sepsis trials and clinical care.